Hppcn Interaction Of Membrane Protein Screening

The mammalian liver possesses a remarkable capacity of regeneration in response to a variety of stimuli. Under normal circumstances, the human liver initiates regeneration within 3 days and has reached its original size by 3-6 months. Liver regeneration has been confirmed to be a very complex process, and hundreds of substances participate in it. The molecular mechanism of liver regeneration is always one of hot areas, which was concerned by many researchers in last several decades.Recently, a cytokine stimulating the hepatocyte proliferation specifically was isolated from the extract of weanling calf liver by our lab and was named HPPCn. And its humanized counterpart was cloned from the cDNA library of human fetal liver. The bioactivity analysis showed that recombinant human HPPCn protein presented the specific stimulation activity on DNA synthesis and cytodieresis of primary cultured rat hepatocyte, human normal liver cell line L02 and human liver cancer cell line SMMC-7721, and hepatectomized mice liver. Experiments in vivo proved the protective effects of rhHPPCn on the acute liver injury and liver fibrosis induced by ethanol and CCl4.The sequence analysis showed that the homogenization of HPPCn with the members of leucine-rich acidic nuclear protein (LANP) family was beyond 70 % and the conserved and special domains were just the same as the other family members of LANP: 1. The N-terminal repeat sequence including 25 leucine residues which was named leucine rich-repeats (LRRs), conformed the characteristic boot-shaped construction withβ-αunits, facilitating the protein-protein interaction; 2. The C-terminal 169-230 residues constituted a highly repeat sequence with GLUs and ASPs which was named acidic tail domain. This domain enabled LANPs to bind chromosome in the nucleus and regulate transcription accordingly. The LANP family members as a kind of multifunctional nucleoprotein involved in various biologic processes including cell proliferation, differentiation and apoptosis and tumor-inhibiting.The protein structure analysis showed that although HPPCn represented the character of nucleoprotein, the typical signal peptide was absent within its structure. The function by which HPPCn exhibits growth factor activity needed to be revealed. A considerable number of nucleoprotein such as HMGB1 and HDGF has been reported to be able to take effects both inside and outside cells. They represent a group of bifunctional cytokines which are active either inside or outside nucleus. The similarity in the structures of HPPCn and these cytokines may produce the similarity in the functions. We wondered that whether HPPCn was a bifunctional cytokine as well. The early study using FITC-tagged rhHPPCn indicated that the rhHPPCn bound to the hepatocyte surface in a plaque-like manner and activated proliferation-related kinases such as SPK, MAPK, STAT-3. Besides, flow cytometry analysis showed either in binding experiment or in competitive inhibition, HPPCn bound to hepatocyte specifically.The present study aims to identify the membrane protein that interacts with HPPCn. His-tagged HPPCn protein was produced and an improved His Pull-down method was used to search the membrane protein which interacted with HPPCn. The interaction between them was confirmed by co-immunoprecipitation and cell transfection. The experimental process was detailed as follow:Firstly, prepare His-tagged HPPCn protein and membrane proteins of SMMC-7721 cell line. The His-tagged HPPCn protein was produced using E. coli. engineering strain BL21 containing express vector pET-24a(+)-HPPCn and purified by Q-XL anion-exchange chromatography and His affinity chromatography. The membrane protein of SMMC-7721 was gained applying the sucrose density gradient centrifugation.Secondly, search the membrane protein interacting with HPPCn by improved His Pull-down. SANPAH (a light-sensitive cross-linker), His-tagged HPPCn protein and membrane protein were exposed to ultraviolet. The cross linkage between His-tagged HPPCn protein and membrane protein occurred because of the cleavage and reset of chemical bonds. The cross-linked complex was isolated by ultracentrifugation and purified by His affinity chromatography. SDS-PAGE was applied to separate the complex. The specificity was determined by Western blot using anti-His and anti-HPPCn antibodies sequentially. The targeted band was identified by comparing the results from SDS-PAGE and Western blot. The mass spectra analysis was applied to determine the component in the recovered gel band.Finally, validate the interaction betweent HPPCn and keratin 8/18. The distribution of keratin 8/18 on SMMC-7721 surface was examined by cellular immunohistochemistry. The interaction between them was further validated by outside-cell binding experiment and co-immunoprecipitation. To determine the presence of this interaction inside cells, the keratin 8/18 express vector pCNA3.1-K8-IRES-K18 was transferred into NIH-3T3 cell line which responds negatively to HPPCn and the proliferation-promotion and anti-apoptosis activity of HPPCn on the transferred cell line was assessed.The results showed that 0.3 mM was the most appropriate IPTG concentration to induce the expression of HPPCn in the engineering strain. After induction for 4 hours, large amount of His-tagged HPPCn protein was yield, indicating that the construction of E.coli. engineering strain expressing His-tagged HPPCn was successful. The bacteria were lysed by ultra-sonication and the supernatant was purified by Q-XL anion-exchange chromatography and His affinity chromatography. The gray scale scan showed that the protein purity was above 90% which met the requirement of the subsequent experiments. A considerable amount of SMMC-7721 membrane protein was gained by adopting sucrose density gradient centrifugation, establishing the foundation for the evaluation of their interaction. The membrane protein interacting with HPPCn was proved to be keratin 8/18 by improved His Pull-down and mass spectrum. The distribution of keratin 8/18 on the surface of cellular membrane was determined by immunohistochemistry. The interaction between HPPCn and keratin 8/18 was validated by protein binding assay and co-immunoprecipitation. Experiments using keratin 8 stable transfected cell line indicated that HPPCn exhibited proliferation-promotion and anti-apoptosis activity in keratin 8 transfected NIH-3T3 cell line.Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair. The data accumμlated in recent years provide that K8/K18 can provide resistance not only to a mechanical stress but toxic stress and Fas-mediated apoptosis.Clinical dates show that the specific amino acid residues of K8/18 lead to various liver diseases. On the one hand, K8/18 maintain cell shape together with other cytoskeletal elements, integral membrane proteins and motor proteins, on the other hand they form complex signaling platforms and interact with various kinases, forms of toxic stress , apoptosis and various liver disease. Therefore K8/18 has a significant importance in the research of hepatocyte resistance to toxic stress, apoptosis and various liver diseases.In this study the membrane protein that interactive with HPPCn was found by His Pull-down and proved to be keratin 8/18. The interaction between them was validated in vitro by co-immunoprecipitation. The keratin 8/18 eukaryotic expression vector was constructed and transferred into NIH-3T3 cell line which responded negatively to HPPCn. The suppression of HPPCn on the proliferation of NIH-3T3 cells was compromised by the expression of keratin 8 alone.